The long term goal of our research has been to understand the relationship of sperm surface organization and sperm function. In previous work we identified two sperm surface proteins, PH-20 and PH-30, that have a role in fertilization. Here we propose to demonstrate their precise function in fertilization nd relate this function to their domain localization, surface density and lateral mobility on the sperm surface. In Aim I we propose to determine the mechanisms responsible for formation of sperm surface domains during spermatogenesis. We will distinguish between two possible mechanisms: 1) the time of expression of surface proteins during spermatogenesis determines their domain localization; 2) surface proteins contain sorting signals that target them to specific domains. (The sorting signal may be used for initial insertion into the correct domain or removal from the "incorrect" domain). We will also analyze the later steps in creation of the anterior and posterior head domains during sperm maturation in the epididymis. In Aim II we present experiments to obtain direct evidence that PH-20 is a sperm receptor for the zona pellucida. We will also test the effect of PH-20's surface density and lateral mobility on its adhesion activity, define its adhesion partner in the zona and test if PH-20 in another species (mouse) functions in sperm-zona binding. We propose in Aim III to study the function of PH-30 which is comprised of the subunits alpha and beta. We will test two hypotheses: PH-30 alpha functions in sperm-egg fusion and its putative fusion peptide is a required active site; PH-30 beta functions in binding sperm to the egg plasma membrane through its N-terminal disintegrin domain. In addition, we propose to identify PH-30 beta's adhesion partner (probably an integrin) on the egg plasma membrane and to test if PH-30 is activated for its role in fusion during epididymal passage, capacitation and/or the acrosome reaction. The importance of this work is that we will make progress in defining both the cellular and molecular basis of the studied steps in fertilization. Furthermore, PH-20 and PH-30 are both under active investigation as antigens for a birth control vaccine and expanded understanding of their basic biology will be useful in that area.